1. Field of the Invention
This invention in general relates to optical scanning systems and, in particular, to an apparatus and method for calibration of a microarray scanning system.
2. Description of the Prior Art
The use of excitation radiation to produce fluorescence in a series of scanned genetic samples is known. U.S. Pat. No. 5,689,110 issued to Dietz et al., for example, discloses a calibration method and device for a fluorescence spectrometer which uses fluorescence from a homogenous solid state standard as the source of calibration fluorescence. Fluorescent imagers are used to acquire data in experiments that utilize fluorescent labels, or fluorophores, to identify the state of a sample being tested. In some cases the presence of or lack of fluorophores in the sample determines the experimental result. In other cases the fluorophore concentration, which is a function of the intensity of the emission radiation received from the sample, is the measurement of interest and the experimental result can be inferred by measuring the intensity of the detected radiation.
An example of a process that uses fluorophores is the microarray which is a set of experiments utilizing genetic material such as DNA or RNA, bound to a glass substrate. Reference or xe2x80x98targetxe2x80x99 DNA is spotted onto a glass substratexe2x80x94typically a one-by three-inch glass microscope slidexe2x80x94where it chemically binds to the surface. Each spot, or sample, of DNA constitutes a separate experiment. A sample of xe2x80x98probexe2x80x99 DNA or RNA, to which has been added the fluorophore material, is subsequently placed on the target spots on the surface of the substrate and is allowed to hybridize with the target DNA. Excess probe DNA that does not bind with target DNA is removed from the surface of the slide in a subsequent washing process.
The experiments measure the binding affinities between the probe DNA and the target DNA to determine the similarity in molecular structure; complementary molecules have a much greater probability of binding than do unrelated molecules. The fluorophore added to the probe DNA emits a range of radiation energy centered about a wavelength xcexemission when illuminated by incident excitation radiation of a particular, shorter wavelength xcexexitation. The brightness of the emitted radiation, measured by the detection system of a microarray scanning system, is a function of the fluorophore concentration present in the illuminated spot. Because the fluorophore concentration is a function of the binding affinity or likeness of the probe molecule to the target molecule, the brightness of a hybridized spot is an indication of the degree of similarity between the probe DNA and the target DNA present in the hybridized spot. A typical microarray sample may provide for up to tens of thousands of experiments to be performed simultaneously on the probe DNA, thus producing a detailed characterization of a particular gene under investigation.
In a microarray scanning system, the area of interest is usually divided into an array of discrete elements referred to as xe2x80x98pixels.xe2x80x99 Each pixel is illuminated independently as it is being addressed by the scanning system. The optical radiation source is typically a single-wavelength laser device focused down to form an excitation radiation spot of the desired size. Emission radiation is emitted by the illuminated fluorophore in an outward, spherical beam. A portion of this emission beam is collected by an optical system and transmitted to a detection apparatus. In addition to the emitted radiation, some of the incident excitation radiation scattered from the surface of the sample is also collected by the optical system. To minimize the amount of excitation radiation reaching the detector assembly, the optical system may be designed using filtering components, such as dichroic and band-pass filters, to provide discrimination between excitation and emission radiation wavelengths.
In order to obtain accurate information from the scanning of a microarray, it is important to know which fluorophore materials have been used in order to use the correct wavelengths in illuminating the spots and to filter the correct wavelengths of the fluorescent emissions. Furthermore, it is advantageous to excite the fluorophores with a high-intensity excitation beam so as to return the maximum signal to the microarray scanning system detector. However, the intensity of the excitation beam must be kept below the level at which the flurorophore becomes saturated or the sample material may degrade.
Furthermore, analysis of raw data collected by the microarray scanning system must be performed in accordance with protocols that may vary in accordance with experiment parameters. In conventional scanning systems, entry of the scanning and analysis protocols is performed manually. This involves significant operator time and, further, is a source of errors in the scanning and analysis procedure.
The sensitivity of the detection system is a critical parameter in a microarray scanning system. The possible range of fluorescence emission varies enormously between samples and often exceeds the dynamic range of the detection system, causing saturation of signals. The occurrence of saturated signals in a data set makes it impossible to quantify the fluorophore brightness emitted from the hybridized spots exhibiting saturation.
In a conventional microarray scanning system, sensitivity adjustment of the detection system is an iterative procedure. The user performs a partial scan using a particular channel of the system, views the image, and adjusts the excitation radiation power and/or the gain of the detector system accordingly such that the optimal range of sensitivity lies within the dynamic range of the detection system. This process is time consuming for the user and, further, degrades the experimental samples by a process of photobleaching the fluorescently-tagged spots on the substrate.
While the relevant art provides iterative procedures for calibration of microarray scanning systems, there remains a need for improvements that offer advantages and capabilities not found in presently available methods of calibration, and it is a primary object of this invention to provide such improvements.
It is another object of the present invention to provide a automatic method of calibrating a microarray scanning system.
It is a further object of the present invention to provide such a calibration method which is performed without damage to the sample microarray.
It is yet another object of the present invention to provide a microarray sample configuration which includes an automatic calibration feature.
Other objects of the invention will be obvious, in part, and, in part, will become apparent when reading the detailed description to follow.
In accordance with the present invention a series of dilution spots is imprinted on a microarray sample which includes an array of genetic material samples containing one or more fluorophores. A microarray scanning system, which includes an excitation radiation source, a detection system, and a computational device, is used to analyze the fluorophores in the genetic material samples. Automatic calibration adjustment of either or both the detection system and the excitation radiation source is achieved by i) irradiating the dilution spots with the source of excitation radiation; ii) detecting emission radiation produced by the dilution spot fluorophore material in response to the irradiation; iii) deriving a series of brightness readings corresponding to the levels of emission radiation detected at corresponding dilution spots; iv) analyzing the brightness readings with the computational device to obtain a fluorophore brightness characteristic as a function of fluorophore concentration; and v) adjusting the sensitivity of the detection system and/or the intensity level of the source of excitation radiation in accordance with the fluorophore brightness characteristic.